P1344: SILK BONE MARROW-LIKE TISSUE MODELS FOR PRODUCING ERYTHROCYTES EX VIVO
Authors: Christian DI Buduo, Francesca Careddu, Marco Lunghi, Virginia Camilotto, Claudia Del Fante, Luca Malcovati, Alessandra Balduini
Journal: HemaSphere (2023)

Topic: 23. Hematopoiesis, stem cells and microenvironment Background: Cellular, chemical, and physical elements define a specialized three-dimensional (3D) bone marrow niche that regulates lineage commitment, and the release of mature blood cells into the bloodstream. Leading studies point to the erythroblastic islands as the core of erythropoiesis. The extracorporeal generation of physiologically relevant models of the bone marrow is central to multiple healthcare challenges including studying the mechanisms of human erythropoiesis in physiological and pathological conditions, drug screening at an individual scale, and production of red blood cells on-demand. Different bioengineering techniques have been developed, aiming at the mass production of erythrocytes in vitro. Tough, extrapolating the 3D structure and function of the erythroblastic islands is challenging. Aims: We have pioneered bone marrow tissue engineering by using silk fibroin, derived from Bombyx mori silkworm cocoons, to provide solutions for human platelet production. Here, we aimed at exploiting this technology to manufacture innovative 3D silk-based bone marrow niches and flow chambers to direct the generation of fully mature human erythrocytes ex vivo. Methods: We developed an all-natural tissue model consisting of silk fibroin and extracellular matrix proteins. A porous spongy scaffold was enclosed in a flow chamber. The flowthrough was used to provide a bespoke cocktail of cytokines and nutrients in combination with increasing concentrations of transferrin. The system was cultured for up to three weeks with hematopoietic stem and progenitor cells (HSPCs) from human-mobilized peripheral blood, upon informed consent and in accordance with the Declaration of Helsinki. Results: After 1 week of culture, flow cytometry analysis demonstrated the gradual loss of stem-associated markers, and the acquisition of the lineage-specific markers CD71 (transferrin receptor), CD36 (a transmembrane scavenger receptor), and CD235a (glycophorin-α). The analysis of the spatial distribution of cells within the silk scaffold, by real-time imaging, demonstrated the spontaneous development of cellular nests, resembling the physiological structure of the erythroblastic islands predominantly aggregated around and/or next to co-cultured CD68+ macrophages. After 2 weeks, the population turned to a CD36neg CD71low CD235ahigh profile, indicative of terminal erythroblast maturation. Starting from week 3, fully mature CD36neg C71neg CD235ahigh erythrocytes were mainly localized at the periphery of the erythroblastic islands, in close contact with macrophages. Dynamic perfusion of the system at physiologically relevant shear stress allowed the collection of enucleated CD235ahigh erythrocytes. Cells were fully hemoglobinized and expressed both α- and β-globin. May-Grunwald Giemsa staining revealed that the size, shape, and color of cultured erythrocytes were comparable to donor cells. Summary/Conclusion: Our data demonstrated that the spatiotemporal control of the dynamics of cell-cell and cell-matrix interaction, together, with controllable shear forces, within a 3D physiologically relevant microenvironment, may be the key to guiding efficient erythropoiesis ex vivo. The silk bone marrow model represents a unique sustainable resource to gain insights into mechanisms of human erythropoiesis, and advance basic knowledge to robust clinical translation. Keywords: Erythrocyte, Bone Marrow, Erythropoieisis